Human carbonic anhydrase ii with increased physical stability

Abstract

An isolated polypeptide having carbonic anhydrase activity, the sequence of which corresponds to modified human carbonic anhydrase II is described. The isolated polypeptide comprises the mutations A23C, S99C, L202C, C205S and V241 C and the polypeptide has increased physical stability compared to wild type carbonic anhydrase II. Further, the polypeptide comprises disulfide bridges between C23 and C202 and / or between C99 and C241.

Description

TECHNICAL FIELD OF THE INVENTION[0001]The present invention relates to an engineered variant of the enzyme human carbonic anhydrase II with increased physical stability as defined by increased thermodynamic, thermal and kinetic stability as compared to the wild type enzyme. The present invention also relates to a method of increasing the physical stability of carbonic anhydrases. Furthermore, the invention relates to the use of said enzyme in any technical application used for CO2 extraction from a medium. Furthermore, the present invention also relates to isolated polynucleotides encoding the polypeptide as well as isolated polypeptides. The invention also relates to nucleic acid constructs and vectors comprising the polynucleotides.BACKGROUND ART[0002]Carbonic anhydrases (CA, EC 4.2.2.1) is a group of enzymes that catalyzes the reversible reaction of carbon dioxide and water into bicarbonate and proton according to:CO2+H2O⇄HCO3−+H+Carbonic anhydrases are widely distributed through...

AI Technical Summary

Benefits of technology

There is a need for an improved carbonic anhydrase that can be used in a bioreactor to capture CO2, as there are no naturally occurring carbonic anhydrases that meet all the requirements needed for a successful bioreactor. The present invention provides an improved carbonic anhydrase that has increased physical stability and a high catalytic activity compared to wild type carbonic anhydrase. The improved carbonic anhydrase has a combination of two stabilizing disulfide bridges at the same positions in humans. These disulfide bridges increase the stability of the carbonic anhydrase and make it more suitable for use in an enzyme-based bioreactor.

Problems solved by technology

However, most of these operate at extreme pressure or temperature and use harmful chemical compounds and still consume high amounts of energy at low efficiency.

However, since the operational conditions such as temperature, pH and chemical composition of the solution etc can vary widely depending on application, neither of these processes is of any value if the necessary carbonic anhydrase catalyst is not stable enough to function at the operational conditions or have long enough life time to be economically viable.

Unfortunately, since there are no organisms living under the conditions that can prevail in a CO2-capturing bioreactor, nature has not provided us with a carbonic anhydrase with the desired stability or efficiency.

Another problem associated with the use of γ-carbonic anhydrase from Methanosarcina thermophila is that to obtain the most active form of the enzyme (Fe2+-Cam) it needs to be produced anaerobically and to be protected from air during purification and use.

One bottleneck in the application of enzymes in industrial processes is that in order to be active, enzymes and other proteins must keep a highly ordered and folded structure.

Thus, it is not possible to deduce the melting point of a protein by simply determine its thermodynamic stability at ambient temperature or vice versa.

That is, at ambient temperatures a protein can have a relatively low thermodynamic stability and still prove to have a relatively high melting point.

However, if this route is no longer accessible, they will be forced to fold via an alternative route that has folding intermediates and transition states of higher energy.

Hence, a high kinetic stability will lead to slow unfolding and a long lifetime of the protein.

However, most methods to stabilize the folded state rely on strengthening local interactions that are only formed once the protein is folded and few will substantially affect the folding route and hence the kinetic stability.

Furthermore, because of the often hundreds of amino acids to vary and the thousands of interactions within the protein and between the protein and the surroundings, it is very difficult to simply examine the structure and pinpoint what to change in order to increase the stability.

Although this method has a limited rate of success since the replacement of the wild type amino acid and the introduction of a disulfide bridge will often lead to loss of favorable interactions or strain in the folded state, it will lead to a larger thermodynamic stability (ΔGFU) if the folded state is unaffected (See FIG. 4).

Firstly, the introduction of even a single stabilizing disulfide bond is challenging, since often what is gained in energy difference by decreased entropy of the unfolded state is often also lost in enthalpic energy in the folded state, because of lost non-covalent interactions, or strain introduced into the structure so that the ΔGFU of the engineered protein is the same or even less than that of the wild type protein (i.e. thermodynamically destabilized).

Thus, introducing two or more disulfide bridges might increase or decrease the stability of the protein.

Secondly, with two disulfide bridges present, the folding pathway of the protein could be blocked, so that the protein is no longer able to fold into its native active form.

Thirdly, when more than two cysteines are introduced in a protein there is a high risk that the cysteines make disulfide bonds with the wrong partner during synthesis or folding.

This will always lead to an inactive protein as it will not be able to find its folded active conformation.

This is also especially important during production of heterologous (e.g. mammalian) proteins with multiple disulfide bonds in recombinant systems (e.g. bacteria) as the formation of correct or native disulfide bonds in such systems is very inefficient, often leading to low yield of production of functional enzymes.

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